The nature and assembly of basement membrane constituents namely IV collagen, laminin and heparan sulphate proteoglycan were studied using a variety of in vitro binding assays. These basement membrane macromolecules were isolated from the EHS tumor grown in C57 black mice. Protease-derived fragments of laminin and IV collagen were characterized by rotary shadowing electron microscopy. The domains required for binding of laminin and IV collagen were identified. Laminin is a cross-shaped molecule with three equal short arms and one long arm. The cell binding region of laminin was also identified and found to reside at the intersection of the three short arms. The carbohydrate composition of laminin was obtained and the distribution of sugars on the long and short arms of laminin molecule was studied. Type IV collagen is a rope-like structure (360 nm) with a globular domain at the carboxyterminal end and a disulphide-rich amino terminal end. A major binding site for laminin is identified at about 100 nm away from the globular end of type IV collagen. The binding domain on laminin for its receptor has now been isolated using protease treatment of laminin. A monoclonal antibody is shown to recognize this domain and block the binding of laminin to the receptor. A complete model has been developed for the orientation of the cell surface laminin receptor, laminin itself, and type IV collagen in the basement membrane.